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• W2967360687 abstract "Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied. We found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently. Comparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs." @default.
• W2967360687 created "2019-08-22" @default.
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• W2967360687 date "2019-08-09" @default.
• W2967360687 modified "2023-09-25" @default.
• W2967360687 title "Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems" @default.
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• W2967360687 doi "https://doi.org/10.1186/s12985-019-1212-y" @default.
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